Mutational analysis of the tRNA3Lys/HIV-1 RNA (primer/template) complex.
Retroviruses use a specific tRNA, whose 3' end is complementary to the 18 nucleotides of the primer binding site (PBS), to prime reverse transcription. Previous work has shown that initiation of HIV-1 reverse transcription is a specific process, in contrast with the subsequent elongation phase. HIV-1 reverse transcriptase (RT) specifically recognizes the complex formed by the viral RNA and tRNA3Lys. We previously proposed a secondary structure model of this complex based on chemical and enzymatic probing. In this model, tRNA3Lysextensively interacts with the genomic RNA. Here, we have combined site-directed mutagenesis and structural probing to test crucial aspects of this model. We found that the complex interactions between tRNA3Lysand HIV-1 RNA, and the intra-molecular rearrangements did not depend on the presence of upstream and downstream viral sequences. Indeed, a short RNA template, encompassing nucleotides 123-217 of the HIV-1 Mal genome, was able, together with the primer tRNA, to adopt the same structure as longer viral RNA fragments. This model primer/template is thus amenable to detailed structural and functional studies. The probing data obtained on the tRNA3Lys/mutant viral RNA complexes support the previously proposed model. Furthermore, they indicate that destroying the complementarity between the anticodon of tRNA3Lysand the so-called viral 'A-rich loop' destabilizes all four helices of the extended tRNA3Lys/HIV-1 RNA interactions.
Isel
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Unité Propre de Recherche No. 9002 du Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex, France.
Keith
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Ehresmann
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B
Ehresmann
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Marquet
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R
D000926:Anticodon / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*
D000926:Anticodon; D017931:DNA Primers; D012357:RNA, Transfer, Lys; D012367:RNA, Viral